Ocelots (Leopardus pardalis) in the United States currently exhibit low levels of genetic diversity. One hypothesis for this observation is that habitat fragmentation, resulting from human induced changes in the landscape during the 20(th) century, created island populations with highly reduced gene flow and increased genetic drift and inbreeding. In an effort to investigate this, we used a portion of the mitochondrial control region and 11 autosomal microsatellite loci to examine historical levels of genetic diversity and infer temporal changes in ocelot populations between 1853 and 2005. Levels of genetic diversity were higher in historical ocelot populations than in extant populations from Texas. The earliest documented loss of mitochondrial haplotype diversity occurred at Laguna Atascosa National Wildlife Refuge. The second extant population inhabiting private lands in Willacy County retained higher levels of genetic diversity through the 1990s, but subsequently lost diversity over the next decade. A similar pattern was observed for autosomal microsatellite loci. This supports the argument that low levels of genetic diversity in Texas are related to human induced population reductions and fragmentation, both of which threaten the remaining ocelots in the United States. At this time, the best means of mitigating the continued erosion of genetic variation are translocation of individuals either from larger populations in Mexico to Texas, or between the Texas populations.